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Fusion protein of single antibody-interleukin 2, its preparation and use

A technology of interleukin and fusion protein, which is applied in the field of application of genetic engineering to prepare the fusion protein, the fusion protein of monoclonal antibody for tumor necrosis treatment and human interleukin 2, and diseases, which can solve adverse reactions, affect the therapeutic effect, lack of specificity

Inactive Publication Date: 2008-05-21
SHANGHAI MEDIPHARM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These cytokines have been sold on the market for many years and have shown unique therapeutic effects. The disadvantages are: short half-life in vivo, lack of specificity
Among them, the three types of antibodies, mouse-derived antibodies, chimeric antibodies or humanized antibodies, contain more or less mouse-derived protein components, which can cause HAMA or HACA reactions when entering the human body, affect the therapeutic effect and may cause serious adverse reactions
[0006] However, fusion of tumor-specific antibodies (especially fully human antibodies) with interleukin-2 to form highly specific and highly tumor-killing fusion proteins has not been performed so far

Method used

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  • Fusion protein of single antibody-interleukin 2, its preparation and use
  • Fusion protein of single antibody-interleukin 2, its preparation and use
  • Fusion protein of single antibody-interleukin 2, its preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Construction of high expression cell line expressing rhTNT-IL2 fusion protein

[0077] (a) Preparation of mouse hybridoma cell line NSO-TNT-1 cells

[0078] Immune mice with tumor cell nucleus extracts, then extract and prepare mouse B cell single cell suspensions, fuse with mouse myeloma cells NS-1 through cell fusion and selective culture techniques, continue to repeatedly culture and screen, and use cell immunization The hybridoma cell line NSO-TNT-1, which highly expresses the mouse TNT monoclonal antibody, was screened and stored at low temperature.

[0079] In the following examples, the chimeric tumor necrosis therapeutic monoclonal antibody TNT-1 expressed by the NSO-TNT-1 cell line was used as the experimental positive control of the antibody component of the fusion protein rhTNT-IL2.

[0080] (b) Construction of mouse myeloma cell line NSO-TNT-IL2

[0081] see figure 1 .

[0082] The whole construction process is divided into two steps. Firstly, a positive...

Embodiment 2

[0093] Expression and purification of rhTNT-IL2 fusion protein

[0094] (a) expression

[0095] Take the frozen mouse myeloma cell line NSO-TNT-IL2 (about 1×10 7 cells), recover at 37°C for 2-3 minutes, transfer the cells in the cryopreservation tube to a 15ml centrifuge tube containing 10ml of selective medium, centrifuge the suspended cells at 1500rpm for 10 minutes, discard the supernatant, and place Centrifuge the pelleted cells and inoculate them in the selection medium to start cell expansion culture until the 3L rotary culture flask, the number of cells>2.0×10 6 cells / ml.

[0096] Large-scale culture expression method:

[0097] 30L of culture medium was filtered through a filter device with a pore size of 0.2 μm, and directly filtered into a sterile bioreactor, and the temperature was maintained at 37±0.5°C. All rhTNT-IL2 cells in 3L spinner flasks were transferred to the bioreactor using a peristaltic pump. Inoculation was performed 36 hours after the medium was f...

Embodiment 3

[0113] Detection of rhTNT-IL2 fusion protein

[0114] (a) IL2 titer determination

[0115] Performed with conventional CTLL-2-dependent cell line / MTT colorimetry. Methods as below:

[0116] (1) Preparation of cell suspension: take a sufficient amount of CTLL-2 cells (ATCC TIB-214) to collect by centrifugation, wash 3 times with basal solution (RPMI1640+10% calf serum), and then resuspend in basal culture medium, Formulated to 5x 10 5 / ml of cell suspension and stored at 37°C.

[0117] (2) Preparation of standard solution: Take 1 standard product (2000IU) and dissolve it according to the instruction manual, then dilute to 200IU / ml with basic culture solution.

[0118] (3) The sample to be tested (stock solution, according to 1×10 6 IU / mg calculation) was diluted to 200IU / ml with basal culture medium.

[0119] (4) In the 96-well cell culture plate, the prepared standard solution (200IU / ml) and the sample (200IU / ml) solution are continuously diluted in a doubling ratio to m...

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Abstract

This invention has disclosed a kind of amalgamation protein of recombinant human tumor necrosis therapy monoclonal antibody (rhTNT) and interleukin 2 (IL2), and the process of coding DNA sequence of this amalgamation protein, building eukaryocyte expression carrier of this amalgamation protein, filtering and preserving cell line of stabilising and expressing highly the amalgamation protein, and purifying the amalgamation protein; And medicine compound contained the amalgamation protein. This amalgamation protein has broad spectrum treatment with anti-tumor.

Description

technical field [0001] The invention relates to the fields of DNA recombination technology and medicine. More specifically, the present invention relates to a fusion protein of tumor necrosis therapeutic monoclonal antibody (rhTNT) and human interleukin 2 (hereinafter referred to as IL2), a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, and a host containing the vector Cells, a process for preparing the fusion protein by genetic engineering, and the application of the fusion protein in treating tumors and other diseases. Background technique [0002] The most important of the current tumor treatment options are surgery, chemotherapy and radiotherapy. Although these three conventional therapies can greatly prolong the survival period of tumor patients, tumor patients still die from recurrence or metastasis, or die from infection or hemorrhage secondary to myelosuppression caused by chemotherapy and radiotherapy. To this end, people continue ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/20A61K39/395A61P35/00
Inventor 鞠佃文陶群叶丹张昕泂傅清
Owner SHANGHAI MEDIPHARM BIOTECH
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